1. The Field of the Invention
This invention relates to cellular support media and to methods by which to prepare cellular support: media for maintaining and supporting either a primary or established cellular line. More particularly, the present invention relates to media and methods that contain bovine calf serum to support either primary or established cellular lines, particularly hybridoma cellular lines, or for use in the production of diagnostic or therapeutic biologicals.
2. The Background Art
It is well-known that animal and plant cells may be grown in vitro in liquid culture media, i.e., tissue culture media. Such media usually contain a wide array of different components, including various nutrients and salts that promote the maximum growth of the cultured cells. Among the most important of these different components are the growth support factors found in the culture medium.
Growth support factors are specific organic compounds that are required in very small amounts and often cannot be synthesized by the cell. Substances frequently serving as growth support factors are vitamins, amino acids, purines, and pyrimidines and peptides, steroids, and proteins. Some organisms are able to synthesize all of these compounds, whereas others require the addition of one or more to the culture medium.
Cells grown in vitro by tissue culture methods are used for many different purposes; for example, for the production of enzymes, secondary byproducts; or for the general testing of drugs, carcinogenic agents, and the like. In vitro growth of animal cells lines has recently acquired new relevance with the development of cell fusion, and the preparation of hybridomas and their associated products, e.g., monoclonal antibodies.
Cells can be obtained from lines that can be characterized as either primary or established. The formation of a cell line from a primary culture implies: (1) an increase in total cell number over several generations, (2) that cells or cell lineages with similar high growth capacity will predominate, and (3) a degree of uniformity in the cell population will result. The line may be characterized, and those characteristics will apply for most of its finite life span. The derivation of "established" (or "continuous") cell lines usually implies a phenotypic change or transformation.
Hybridoma cell lines are artificially created to be employed in immunological studies and the production of diagnostic or therapeutic biologicals production., A typical immune response results in the production of a broad spectrum of antibodies of varying affinities for any given antigen. The antibodies directed toward a particular determinant will represent but a small proportion of the total antibody pool. If one could isolate a hybrid clone of B cells that are responsible for making a particular antibody of interest, a source of monospecific or monoclonal antibody would be available.
Techniques are now available for isolating and growing single hybrid B-lymphocytes for indefinite periods of time. This procedure, called the "hybridoma technique," combines the unlimited division properties of a cancer cell with the monoclonal antibody production of a single B-lymphocyte. In practice, myeloma cells are fused with a pool of B cells removed from a mouse previously immunized with the antigen of interest; agents (such as polyethylene glycol) are added to the mixture to promote fusion.
Since myeloma cells are themselves B-lymphocytes, a variant myeloma cell line that has lost the ability to make immunoglobulin is routinely employed in hybridoma production. After fusing, the heterokaryotic hybrid cell undergoes a nuclear fusion and eventually results in antibody producing cells that can be selectively cloned and subsequently injected into an animal, where it grows as a myeloma tumor secreting monoclonal antibodies. The immunoglobulin molecules produced by a hybridoma are characteristic of the normal B cell to which the malignant cell was fused. The resulting hybridoma population is screened using various immunological and biochemical techniques to identify the clone producing the monoclonal antibody of interest.
The art has long established that one of the essential components for tissue culture media is animal serum, i.e,, fluid taken from clotted blood (as opposed to plasma which is obtained via centrifugation of blood prior to clotting). Most preferably, fetal bovine serum is employed for tissue culture media. This type of serum lacks high concentrations of the components that inhibit cell growth, and contains undefined factors that support cell growth in vitro.
Fetal bovine sera, as opposed to other types of sera, are employed in culture media because organisms, from which sera are obtained, at an early age have less experienced immune responses. This is possible since fetal bovine sera comes from animals taken from the in utero state where their systems have not come into contact with high levels of contaminants. Fetal bovine serum contains factors that promote development of the fetus and are thereby often necessary for a cell's maintenance and support in vitro.
The use of fetal bovine serum, however, is troubled by a lack of sufficient supply. Because fetal bovine serum can only be derived from pregnant cows, the sources from which the serum can be obtained are limited. Moreover, the number of available cows can vary depending upon activity in the meat industry and the open markets wherein the demand for beef can fluctuate without warning.
The production of an alternative fetal bovine serum to meet the problems with supply is possible. Production, however, would require that the alternative serum be made from the constituent substances which comprise fetal bovine serum. This procedure is obviously expensive and time consuming. Therefore, the increasing demand for the limited supplies of fetal bovine serum forces researchers to look for other alternatives for fetal bovine serum available in great quantities.
The use of fetal bovine serum is also troubled by the poor characterization of its ingredients. Typically, the cellular lines grown in the fetal bovine serum are employed for testing and it may be necessary to identify the factors that support the cellular growth. Identification of the factors can take a long period of time and quite a bit of effort to perform several assays on the fetal bovine serum. This is important to scientists conducting tests with media employing the sera; use of sera whose ingredients are better characterized will promote research and valuable testing.
In addition, the use of fetal bovine serum is troubled due to the undefined amount of time the serum remains stable while stored. Fetal bovine serum is comprised of various ingredients (such as vitamins and proteins) that can become inactivated over time. Therefore, a large amount of the serum can not be kept over extended periods of time and, therefore, the serum must constantly be reordered, which as previously mentioned, may not be possible since the serum supply is often limited.
Furthermore, the use of fetal bovine serum is troubled by the costs for this type of serum which can preclude the existence of cell culture technology because of the expenses involved in the acquisition of fetal bovine serum. An alternative serum available at a fraction of the cost of fetal bovine serum would be in great demand. This factor increases the emphasis society has placed on researchers to find a cost-effective alternative for fetal bovine serum.
This is particularly important to the development of hybridoma cellular lines. Hybridoma cellular lines are artificially created to be employed in immunological studies and the production of diagnostic or therapeutic biologicals. High costs for fetal serum (due to shortages in supply, short time the serum remains stable, and the like) may preclude the existence of hybridoma technology due to economic considerations associated with fetal bovine serum with respect to hybridoma cellular lines.
A need, therefore, exists for media used to maintain or promote primary or established cellular lines that can meet the existing and anticipated increasing future demand for growth media. The media should be available at a low price so that the economically feasible growth of cellular lines that utilize the media is promoted. This is particularly true of hybridoma cellular lines that are increasingly being employed to produce diagnostic and therapeutic biologicals. Likewise, a need exists to decrease the cost of fetal bovine serum-containing media in order to enable the economically feasible growth of hybridoma cells.
Moreover, the cost of reagents employed to make up media used to maintain and promote cellular lines needs to be lower. If reagents can be purchased at a lower price, the cost of the media comprising the reagents can likewise be purchased at a lower price.
Similarly, a need exists to provide a serum-containing media that will remain in a stable condition over an extended period of time and will not require replacement if stored for extended periods of time. Frequent replacement of sera subject to rapid degradation necessarily results in higher costs to the individual employing the sera.
A need further exists to provide serum-containing media that does not suffer from poor characterization of its ingredients. Scientists can proceed with various tests knowing the precise components comprising their testing media.